Differentiated human liver cell cultures and their use in bioartificial liver systems

ABSTRACT

Human hepatocyte cell cultures and their use in bioreactors and bioartificial liver (BAL) systems are provided. The cells have constitutive liver-specific metabolic activity resembling that of freshly isolated human hepatocytes. The hepatocyte cells and BAL systems can be used to treat patients suffering from acute liver failure, end-stage liver disease, or acute-on-chronic liver disease.

FIELD OF THE INVENTION

The present invention concerns the field of bioartificial liver (BAL)systems. Such BAL systems are used to treat subjects suffering fromliver failure to temporarily compensate for loss of hepatocellularfunction and generally comprise a bioreactor loaded with functionalliver cells. The present invention, in particular, concerns human livercells that are particularly suitable for use in these BAL systems. Thepresent invention furthermore provides new BAL systems, configured toallow for these human liver cells to be utilized to their fullpotential. Furthermore, the present invention concerns the use of theaforementioned human liver cells and the aforementioned BAL systems fortreating subjects suffering from a condition resulting in loss ofhepatocellular function, such as acute liver failure, end-stage liverdisease or acute-on-chronic liver disease.

BACKGROUND OF THE INVENTION

Chronic liver disease is responsible for over 1.4 million disabilityadjusted life years annually and ranks in the United States among thetop 7 disease-related causes of death between the age of 25 and 64years. For end-stage liver failure, orthotopic liver transplantationremains the current treatment of choice. However, patients sufferingfrom acute or acute-on-chronic liver failure may benefit from temporaryextracorporeal artificial liver support used to tide them over untiltransplantation or to allow regeneration of their own liver to occur.

Bioartificial liver (BAL) systems seem to be a promising solution forthis purpose. These systems allow for extracorporeal blood (plasma)treatment relying on functional liver cell cultures for detoxificationand synthetic function. Over the last 2. decades, many BAL systems havebeen devised, only some of which systems have been applied in a clinical(pilot) setting. BALs are based on a bioreactor facilitating functionalliver cells and can be employed to compensate for the loss ofhepatocellular function by perfusing the reactor with the subject'sblood plasma. Some BAL systems comprise a bioreactor in combination witha non-biological detoxification modality, such as a charcoal column or abilirubin column (so called hybrid systems). For a more completeunderstanding of existing BAL technology, Park and Lee¹ or Sgroi et al.²may be referred to, providing comprehensive overviews of the differenttypes of reactors and the different system configurations developed byvarious groups up to 2009.

The cell source employed in the BAL bioreactor should exhibit highhepatic functionality. Although the pathogenesis of hepaticencephalopathy (HE), one of the major causes of death of ALF, ismultifactorial, it is generally accepted that hyperammonemia plays acrucial role. Increased plasma ammonia levels are toxic for the brain,contribute to brain edema and stimulate inhibitory neurotransmission. Avery essential liver function is detoxifying blood ammonia, primarily byurea synthesis and secondarily by glutamine synthesis. Urea is non toxicand is rapidly excreted by the kidneys. Glutamine is non-toxic as welland can be used as metabolic substrate by different organs among whichthe intestine. In addition, the cell source should displaydetoxification of other accumulating endogenous toxic compounds mainlythrough the cytochrome P450 system, synthesis of blood proteins andhomeostasis of amino acids, lipids and carbohydrates.

Many liver cell types have been used for preclinical and clinicalapplication of BAL devices. The choice for the optimal cell source forBAL support devices is, however, still a matter of debate. Particularly,the availability, degree of liver specific function, and safety aspectsare ongoing issues. Primary hepatocytes, either allogeneic orxenogeneic, have an excellent function and from that perspective wouldappear to be the favourite cell sources for BAL application.

However, mature porcine hepatocytes are not attractive for clinicalapplication because of the obvious risks and objections related tomethods of treatment of human beings involving xenotransplantation.

Mature human hepatocytes, on the other hand, are scarce, as theirsources are limited to either discarded donor livers or small partsobtained during liver resections,

Human hepatic cell lines have the advantage of an infinite proliferationcapacity and can potentially serve as a stable cell source. In addition,the use of such cell lines from human origin would effectively avoid thexenotransplantation associated risks and objections. From theseperspectives human hepatocyte cell lines seem to offer the mostpromising starting point for successful BAL development. Notsurprisingly therefore, a lot of effort has been invested in finding anddeveloping human hepatocyte cell lines having the required hepaticfunctionalities. So far, these efforts have only resulted in verylimited success though.

The potential of a human fetal liver cell line cBAL111 for applicationin BAL systems has been tested and described by Poyck et al.³. Poyck etal. reported that cBAL111 eliminated ammonia at a rate up to 49% of thatin primary porcine hepatocytes (PPH), despite a low urea production.Their synthetic functions (albumin production: 6%) and detoxificationfunctions (lidocaine elimination: 1%) were to be low.

Uses of HepG2 cells or genetically modified variants thereof, in severalBAL systems have been described by e.g. Nyberg et al.⁴, Wang et al.⁵,Enosawa et al.⁶, and Takahashi et al.⁷ .It is now well-established thatthe urea cycle is not maintained in HepG2 cells resulting in the lack ofammonia detoxification via this route. Gene expression data from HepG2cells reveals limited expression of three urea cycle genes CarbamoylPhosphate Synthase I (CPS),

Arginosuccinate Synthetase (ASS) and Arginosuccinate Lyase, whereas noexpression of Ornithine Transcarbamylase can be established.

According to Mavri-Damelin et al.⁸, a HepG2 sub-clone, designated C3A,nevertheless had been found to produce urea. Their research confirmedthat gene expression of ornithine transcarbamylase (OTC) and arginase I(Arg I), were completely absent. Arginase II (Arg II) mRNA and proteinwas expressed at high levels in C3A cells though and was inhibited byN^(ω)-hydroxy-nor-L-arginine, which prevented urea production, therebyindicating a urea cycle-independent pathway. The authors conclude thatthe urea cycle is non-functional in C3A cells, and that these cellstherefore cannot provide ammonia detoxification in a BAL system viaurea. The authors note that this emphasizes the continued requirementfor developing a component capable of a full repertoire of liverfunction, including an intact urea cycle to detoxify ammonia.

Kosuge et al.⁹ reported that in gene expression profiles of bioreactorgrown FLC-4, FLC-5 and FLC-7 cells some genes for liver functions wereexpressed at a level similar to that in normal liver, although none ofthe cell lines expressed the complete set of genes encoding ammoniummetabolising enzymes or cytochrome P450 species. The use of a BAL systemloaded with FLC-4 cells for treating pigs with hepatic dysfunction wasreported by Kanai et al.¹⁰. However, the FL-4 cells were defect in theirurea cycle.

Saito et al.¹¹ reported the development of a BAL system comprising FLC-5cells. To assess hepatocyte function of the cells incubated in the BAL,expression of urea cycle and albumin synthesis enzymes were studied. Theauthors reported the presence of urea in the medium and an increase inthe expression of urea cycle enzymes ASS and Arg I for the BAL incubatedcells, whereas albumin synthesis had decreased. All in all, no FLC cellline has been obtained so far providing complete metabolic functionalitythat would render it suitable for clinical BAL application.

US 2005/0064594 discloses a liver cell line, designated HepaRG, whichapproximates human hepatocytes in cytochrome P450 3A activity (CYP3A4),which is a key factor in detoxification, after culturing for 14 days inHepaRG medium with 2% of dimethylsulfoxide (DMSO) after an initial14-days proliferation phase without the addition of DMSO. In thesecultures growth as well as many of the required hepatic functions otherthan CYP3A4, like urea synthesis and albumin synthesis are suppressed.

In summary, each of the human cell lines described above all havecertain detoxification and/or metabolic liver functionalities, whereascertain other, essential, detoxification and/or metabolic liverfunctionalities are substantially reduced compared to those in freshlyisolated human hepatocytes. In particular, until now, it has beenproblematic to acquire cells with a broad spectrum functionality,resembling that of freshly isolated human hepatocytes, to the extentthat they are in fact suitable for successful clinical BAL application.Consequently, so far, it has proven impossible to develop a human cellline based BAL system providing complete hepatic functionality,especially a system providing both detoxification functionality as wellas complete metabolic functionality.

It was the objective of the present inventors to provide adequatesolutions to these short-comings of the prior art cells and BAL systems,thereby bringing successful clinical application a significant stepcloser.

SUMMARY OF THE INVENTION

The present inventors have managed to develop human hepatocyte cell linecultures that display broad-spectrum functionality, exceeding that ofthe cell lines currently known in the art. More in particular cellcultures are provided that display RNA expression profiles and hepaticfunctionality, inter alia, ammonia elimination, urea synthesis, ApoA1production and albumin production, which render them suitable foreffective clinical BAL application.

The present inventors have established that meeting this objectiverequires use of the proper cell line as well as using proper conditionsfor directing differentiation of the cells. More, in particular, it wasfound feasible to direct the differentiation of certain cell lines intothe desired phenotype depending on the presence or absence of certainchemicals in the culture media.

The present inventors furthermore established that the functionality ofthe cell cultures is enhanced by culturing in a system allowing forthree-dimensional growth, e.g. in a bioreactor comprising athree-dimensional solid support.

As will be explained here after in more detail, cell cultures can beobtained in accordance with the invention, having unparalleledbroad-spectrum hepatic functionality.

Thus, the present invention provides cell cultures, a bioreactorcontaining them and BAL systems as well as their uses and their methodsof manufacture. Furthermore, the present invention provides methods ofimproving the functionality of hepatic cell line derived cultures byculturing them in specific culture media and/or under conditionsallowing for three-dimensional growth.

These and other aspects of the invention will be explained andillustrated in more detail in the following description and examples.

DETAILED DESCRIPTION OF THE INVENTION Human hepatocyte cell linecultures

A first aspect of the invention concerns a cell culture comprisingdifferentiated cells from a human hepatocyte cell line in a suitableculture medium, wherein said differentiated cells have constitutivelyliver-specific metabolic activity.

By “cell culture” or “culture”, is normally meant an artificial in vitrosystem containing viable cells, whether quiescent, senescent or(actively) dividing. In a cell culture, cells are grown and maintainedat an appropriate temperature, typically a temperature of 37° C. andunder an atmosphere typically containing oxygen and CO₂. Cultureconditions may vary widely for each cell type though, and variation ofconditions for a particular cell type can result in different phenotypesbeing expressed. The most commonly varied factor in culture systems isthe growth medium. Growth media can vary in concentration of nutrients,growth factors, and the presence of other components. The growth factorsused to supplement media are often derived from animal blood, such ascalf serum.

In accordance with a preferred embodiment of the present invention, themedium containing the differentiated cells can be any medium whereinviability and phenotype of the cells is maintained. This medium in whichthe differentiated cells are contained is not necessarily the same asthe medium(s) employed for the proliferation and/or differentiationstages, which may require specific medium compositions as will beexplained hereafter. Preferably said suitable medium containing thedifferentiated cells at least comprises serum, hormones, growth-factorsand antibiotics. As will be understood by the person skilled in the art,serum free chemically defined media may be used instead withoutdeparting from the scope of the invention. For the reasons explainedbelow, it is also preferred that said medium does not containsubstantial amounts of DMSO, most preferably it is free from DMSO.

As will be known to the skilled person, cells can be provided insuspension or adherent cultures. Some cells naturally live insuspension, without being attached to a surface, such as cells thatexist in the bloodstream. There are also cell lines that have beenmodified to be able to survive in suspension cultures so that they canbe grown to a higher density than adherent conditions would allow.Adherent cells require a surface, such as plastic, which may be coatedwith extracellular matrix components to increase adhesion properties andprovide other signals needed for growth and differentiation. Most cellsderived from solid tissues are adherent. Another type of adherentculture involves growing cells in a three-dimensional environment asopposed to two-dimensional culture dishes. This 3D culture system isbiochemically and physiologically more similar to in vivo tissue. Livercells are typically polarized cells and function optimally when growingunder cell-cell contact conditions in 3D configuration. Therefore, inaccordance with the present invention, it is preferred that the cellculture comprises cells grown in a 3D culture, typically on athree-dimensional support matrix material, as will be explained in moredetail hereafter.

Furthermore, the cell cultures of the present invention are typically ina state of confluence. The term “confluence” as used herein, refers to adensity of cultured cells in which the cells contact one anothercovering most or all of the surfaces available for growth. Duringpre-confluent growth, selected cells behave like regeneratinghepatocytes demonstrating corresponding patterns of regulation of geneexpression. Upon reaching confluence, the cells assume an adultphenotype wherein cell division slows dramatically (typically doublingtime >200 hr).

Human hepatocytes are the cells making up 70-80% of the total cell massof the human liver. Hepatocytes are involved in protein synthesis,protein storage and transformation of carbohydrates, synthesis ofcholesterol, bile salts and phospholipids, and detoxification,modification and excretion of exogenous and endogenous substances.Primary hepatocytes are typically separated from liver by collagenasedigestion. This process creates a suspension of hepatocytes, which canbe cultured and plated for immediate use, optionally cryopreserved.Primary mature human hepatocytes obtained in this way do not proliferatein culture. As mentioned above this effectively renders them unsuitablefor use in BAL systems. The present invention therefore concernshepatocyte cell lines.

The term “cell line”, as used in the context of the present inventionapplies to cells cultured in vitro that have arisen from a primaryculture and are capable of successful subculture. Primary cultures ofdifferentiated mature mammalian cells do not multiply in culture orcease to multiply in culture after a limited number of divisions. Thehepatocyte cell lines according to the present invention are typicallycapable of a significant number of cell divisions. Preferably, thepresent cultures contain cells obtained after at least 20, 30, 40, 50,60, 70, 80, 90, and preferably at least 100 population doublings. Thiscapability is typically found in cells derived from hepatic tumours butit can also be attained by immortalization of primary mature or fetalhepatocytes or in stem cells that might differentiate into hepatocytes.These stem cells may derive from a variety of sources includingembryonic stem cell lines, liver, and other tissues, like pancreas, fat,bone marrow and cord-blood. The capability of the cells according to theinvention to multiply indefinitely advantageously allows for theobtaining of large amounts of cells by multiplication or proliferationof these cells in vitro.

Thus, in a particularly preferred embodiment of the invention a cellculture is provided as defined herein before wherein said cells have ahigh proliferation capacity, but also have the capacity to retain orgain hepatic functionality.

A particularly preferred example of such a cell line is designatedHepaRG as deposited on 5 Apr. 2001 at the Collection Nationale deCultures de Microorganismes, Institut pasteur, under No. 1-2652. Asstated before, this cell line has been described in the prior art, e.g.in US 2005/0064594. This document however teaches to employ a mediumcontaining DMSO to initiate differentiation. The cell cultures thusobtained display mainly liver-specific detoxification functionality. Thepresent inventors have for the first time produced a cell culture basedon HepaRG cells differentiated under different conditions, yielding cellcultures displaying significant metabolic activity, but also syntheticand detoxification properties.

In another particularly preferred embodiment of the invention, a cellculture, is provided, comprising differentiated cells clonally derivedfrom the aforementioned HepaRG cell line, including cells that have beenderived from HepaRG using recombinant DNA techniques.

In other embodiments of the invention, cell lines designated HepG2 (ATCCnumber: HC-8065) and cBAL111 (Deurholt ¹⁹) are used. The presentinventors found that these cell lines can also suitably cultured inaccordance with the methods of the present invention to yield cellcultures displaying significantly enhanced hepatic functionality ascompared to cultures grown under conditions previously described, aswill be illustrated in more detail in the examples. Cellulardifferentiation is the process by which a less specialized cell becomesa more specialized cell type. Differentiation occurs numerous timesduring the development of a multicellular organism as the organismchanges from a single zygote to a complex system of tissues and celltypes. Differentiation can dramatically change a cell's size, shape,membrane potential, metabolic activity, and responsiveness to signals.These changes are largely due to modifications in gene expression; witha few exceptions, cellular differentiation never involves a change inthe genome itself. Each specialized cell type in an organism expresses asubset of all the genes that constitute the genome of that species. Celldifferentiation thus involves a switch from one pattern of geneexpression to another. In accordance with the present invention the term“differentiated cells” is thus used to indicate that the cells of thehuman hepatocyte cell line have been cultured under certainpredetermined conditions in order for them to adopt a certain pattern ofgene expression resulting in a specific phenotype. Differentiallydifferentiated cells are physically distinct entities, as will berecognized by the skilled person. As noted before, in accordance withthe invention, the cells have differentiated such as to displayliver-specific activity, as will be explained in more detail here after.

In the normal functioning liver, the major metabolic functions ofhepatocytes can be divided into three major categories: carbohydratemetabolism, fat metabolism and protein metabolism. Carbohydratemetabolism mainly involves maintenance of normal blood glucose levels.Hepatocytes house many metabolic pathways including glycogenesis,glycogenolyis and gluconeogenesis. Many aspects of lipid metabolism arecarried out predominantly by the liver (although few are unique to theliver). Major examples of the role of the liver in fat metabolisminclude oxidation of triglycerides and synthesis of lipoproteins,cholesterol and phospholipids. The most critical aspects of proteinmetabolism that occur in the liver are deamination and transamination ofamino acids, followed by conversion of the non-nitrogenous part of thosemolecules to glucose or lipids; removal of ammonia from the body bysynthesis of urea; synthesis of non-essential amino acids; and synthesisof most of the plasma proteins. Albumin, the major plasma protein, issynthesized almost exclusively by the liver. Also, the liver synthesizesmany of the clotting factors necessary for blood coagulation andproduces growth factors. Hence, the term “liver metabolic functionality”as used in the context of the present invention, in principleencompasses any one or more of the above functions. However, as notedabove, the present invention, in particular aims at improving themetabolic functionality of the cells with the purpose of treatingsubjects suffering liver failure. In this respect, as will be understoodby the skilled person, some of the above liver metabolic functions aremore critical than others.

In accordance with the invention, liver specific metabolic activity ispreferably characterized, by a functioning urea cycle, preferably by arate of ammonium elimination and/or urea production above a certainpredetermined minimum, such as to render them suitable for practical BALapplication.

Hence, in a preferred embodiment of the invention a cell culture asdefined herein before is provided, characterized by a rate of ammoniaelimination of at least 0.05 μmol/h/mg protein, under suitable cultureconditions. More preferably said rate is at least 0.1 μmol/h/mg protein,most preferably at least 0.2 μmol/h/mg protein. Preferably, the rate ofelimination of ammonia by a preferred cell culture of the invention isat least about 5, 10, 25, 50 or 75% of the rate of primary mature humanhepatocytes under substantially identical in vitro culture conditions.

Furthermore, in a preferred embodiment of the invention a cell cultureas defined herein before is provided, characterized by a rate of ureaproduction of at least 0.003 μmol/h/mg protein, under suitable cultureconditions. More preferably said rate is at least 0.005 μmol/h/mgprotein, most preferably at least 0.010 μmol/h/mg protein. Preferably,the rate of urea production by a preferred cell culture of the inventionis at least about 1, 2, 5, 10, 25, 50 or 75% of the rate of primarymature human hepatocytes under substantially identical in vitro cultureconditions.

Urea may also be produced without elimination of ammonia. In a preferredembodiment of the invention a cell culture as defined herein before isprovided, characterized by a % of urea derived from exogenously addedammonia of at least 10% of the total produced urea, under suitableculture conditions. More preferably said ratio urea formed fromexogenously added ammonia versus total produced urea is at least 15%,most preferably at least 30%.

In addition to the urea cycle, liver specific metabolic activity inaccordance with the present invention also preferably involves otherprotein, carbohydrate and/or lipid metabolic mechanisms.

In accordance with the present invention, the present cell culturespreferably are also characterised by their ability to produce albuminSerum albumin is the most abundant blood plasma protein and is normallyproduced in the (healthy) liver. Serum albumin is important inregulating blood volume by maintaining the oncotic pressure (also knownas colloid osmotic pressure) of the blood compartment and is animportant transporter of many non-water soluble compounds like e.g freefatty acids and bilirubin. As will be shown in more detail in theexamples, a cell culture is provided by the present inventors capable ofproducing albumin at a rate of about 10 ng/h/mg protein. Hence, in apreferred embodiment of the invention a cell culture as defined hereinbefore is provided, characterized by a rate of albumin production of atleast 1 ng/h/mg protein, under suitable culture conditions. Morepreferably said rate is at least 2 ng/h/mg protein, still morepreferably at least 5 ng/h/mg protein and most preferably at least 10ng/h/mg protein. Preferably, the rate of albumin production by apreferred cell culture of the invention is at least about 5, 10, 25, 50or 75% of the rate of primary mature human hepatocytes undersubstantially identical in vitro culture conditions.

In accordance with the present invention, the present cell culturespreferably are also characterised by their ability to produce Apo A1.Apolipoprotein A1 is the major protein component of high densitylipoprotein (HDL) in plasma and promotes cholesterol efflux from tissuesto the liver for excretion. The protein is normally produced in thehealthy liver and intestine. The capability of the present cell culturesto produce Apo A1 is therefore also typically indicative of adequatemetabolic functionality. In addition, ApoA1 has an anti-inflammatoryeffect, which may be beneficial during liver disease. In a preferredembodiment of the invention a cell culture as defined herein before isprovided, characterized by a rate of Apo A1 production of at least 0.002ng/h/mg protein, preferably at least 0.005 ng/h/mg, more preferably atleast 0.01 ng/h/mg, more preferably at least 0.02 ng/h/mg, especially atleast 700, at least 1000, at least 1500 or at least 2000ng/h/mg protein.Preferably, the rate of Apo A1 production by a preferred cell culture ofthe invention is at least about 5, 10, 25, 50 or 75% of the rate ofprimary mature human hepatocytes under substantially identical in vitroculture conditions.

Furthermore, adequate gluconeogenesis by the cells, in particularlactate consumption and glucose production rates are also consideredindicative of liver metabolic functionality. Hence, in a preferredembodiment of the invention a cell culture as defined herein before isprovided, characterized by a rate of lactate consumption of at least0.02 μmol/h/mg protein, under suitable culture conditions. Morepreferably said rate is at least 0.05 μmol/h/mg protein, most preferablyat least 0.1 μmol/h/mg protein. Preferably, the rate of lactateconsumption by a cell culture of the invention is at least about 5, 10,25, 50 or 75% of the rate of primary mature human hepatocytes undersubstantially identical in vitro culture conditions.

Furthermore, in a preferred embodiment, the cells display liver-specificdetoxification activity, typically characterized by a rate of productionof 63-hydroxy-testosterone from testosterone of at least0.5, preferablyat least 1.0, more preferably at least 2.0 nmol/h/mg protein, undersuitable culture conditions. Preferably, the 63-hydroxy-testosteroneproduction rate of the cell culture is at least about 20, 40, 60, 80, or100% of the rate of primary mature human hepatocytes under substantiallyidentical in vitro culture conditions. As will be understood by theskilled person, adequate detoxification functions of the cells are alsocharacterized by the expression of certain genes, coding for the enzymesor proteins involved in these functionalities. In a particularlypreferred embodiment, said differentiated cells having constitutivelyliver-specific metabolic activity are characterized by at least two,preferably three, more preferably all, of the following metabolicparameters:

a rate of ammonia elimination of at least 0.05 μmol/h/mg protein;a rate of urea production of at least 0.003 μmol/h/mg protein;a rate of apoA1 production of at least 15 ng/h/mg protein;a rate of lactate consumption of at least 0.02 μmol/h/mg protein and.a rate of albumin production of at least 1 ng/h/mg protein

In another particularly preferred embodiment, said differentiated cellshaving constitutively liver-specific activity are characterized by atleast three, preferably four, more preferably all, of the followingparameters:

a rate of ammonia elimination of at least 0.05 μmol/h/mg protein;a rate of urea production of at least 0.003 μmol/h/mg protein;at least 10% urea produced from exogenously added ammonia of totalproduced ureaa rate of apoA1 production of at least 700 ng/h/mg protein;a rate of lactate consumption of at least 0.02 μmol/h/mg protein;a rate of albumin production of at least 1 ng/h/mg protein anda rate of 6β-hydroxy-testosterone production of at least 0.5 nmol/h/mgprotein

As will be understood by the skilled person, adequate urea cyclefunctionality as well as other functions of the cells are alsocharacterized by the expression of certain genes, coding for the enzymesor proteins involved in these functionalities. The expression of mostgenes is regulated at the transcription level. Therefore the expressionlevels of specific genes can be determined by reverse-transcriptasepolymerase chain reaction (RT-PCR). As indicated by Hoekstra et al.¹²,mRNA quantification can be carried out by normalization for the 18S rRNAstarting level and subsequently standardized for average mRNA startinglevels of two independent human healthy liver samples.

HNF4 (Hepatocyte Nuclear Factor 4) is a nuclear receptor protein mostlyexpressed in the liver. HNF4 has been found to be a central player inthe hepatocyte nuclear factor network driving hepatic differentiationand function, driving e.g. the expression of proteins involved indetoxification and lipid metabolism and blood proteins, as well as otherhepatocyte specific transcription factors. The present inventors believethat HNF4 expression is indicative of hepatic differentiation. As willbe shown in more detail in the examples, a cell culture is provided bythe present inventors having HNF4 transcript levels of up to about 250%.Hence in a preferred embodiment of the present invention a cell cultureas defined herein before is provided, characterized by transcript levelsof HNF4 of at least 50% of the in vivo level. More preferably, saidtranscript level is at least 100% and most preferably at least 200% ofthat of healthy adult liver.

The pregnane X receptor (PXR), also known as NR1I2 (nuclear receptorsubfamily 1, to group I, member 2), is a nuclear receptor that canup-regulate the expression of proteins involved in the detoxificationand clearance of these substance from the body. One of the primarytargets of PXR activation is the induction of CYP3A4, an important phaseI oxidative enzyme that is responsible for the metabolism of many drugs.The present inventors believe that a significant rate of PXR expressionis indicative of adequate liver-specific detoxification functionality.As will be shown in more detail in the examples, a cell culture isprovided by the present inventors having PXR transcript levels of about70%. Hence in a preferred embodiment of the present invention a cellculture as defined herein before is provided, characterized bytranscript levels of PXR of at least 25% of the in vivo level. Morepreferably, said transcript level is at least 50% and most preferably atleast 80% of that of healthy adult liver.

Carbamoyl phosphate synthetase (CPS) is the enzyme that catalyzes thereaction of ammonia and bicarbonate to produce carbamyl phosphate. Undernormal physiological conditions CPS is the rate limiting enzyme of theurea cycle. During one turn of the urea cycle one nitrogen of urea isderived from ammonia and one from aspartate. The urea cycle is anessential liver specific function to protect the body (especially thebrain) against hyperammonemia. Hence in a preferred embodiment of thepresent invention a cell culture as defined herein before is provided,characterized by transcript levels of carbamoyl phosphate synthetase(CPS) of at least 20% of the in vivo level. More preferably, saidtranscript level is at least 40, 50, 75 or 90% of that of healthy adultliver.

Ornithine transcarbamoylase (OTC) (also called ornithinecarbamoyltransferase) is an enzyme that catalyzes the reaction betweencarbamoyl phosphate (CP) and ornithine (Orn) to form citrulline (Cit)and phosphate (P_(i)). OTC in mammals is located in the mitochondria andis part of the urea cycle. Hence in a preferred embodiment of thepresent invention a cell culture as defined herein before is provided,characterized by transcript levels of Ornithine transcarbamoylase (OTC)of at least 25% of the in vivo level. More preferably, said transcriptlevel is at least 30, 35, 40 or 50% of that of healthy adult liver.

Arginase I (Arg I) is an enzyme that catalyzes the synthesis of ureafrom arginine. Arg I in mammals is located in the cytoplasm and is partof the urea cycle. In an embodiment of the present invention a cellculture as defined herein before is provided, characterized bytranscript levels of Arginase I (Arg I) of at least 15% of the in vivolevel. More preferably, said transcript level is at least 20, 35, 40 or50% of that of healthy adult liver.

Glutamine synthetase (GS) is an enzyme that plays an essential role inthe metabolism of nitrogen by catalyzing the condensation of glutamateand ammonia to form glutamine. As such, significant GS transcript levelsare typically indicative of adequate liver metabolic functionality. Aswill be shown in more detail in the examples, a cell culture is providedby the present inventors having GS transcript levels of about 300%.Hence, in a preferred embodiment of the invention a cell culture asdefined herein before is provided, characterized by transcript levels ofglutamine synthetase (GS) of at least 100% of the in vivo level. Morepreferably, said transcript level is at least 200, 250 or 300% of thatof healthy adult liver.

Cytochrome P450 3A4 (abbreviated CYP3A4), a member of the cytochromeP450 mixed-function oxidase system, is one of the most important enzymesinvolved in the metabolism of xenobiotics in the human body. CYP3A4 isinvolved in the oxidation of the largest range of substrates of all theCYPs. CYP3A4 is also, correspondingly, present in the largest quantityof all the CYPs in the human liver. The present inventors believe that asignificant rate of CYP3A4 expression is indicative of adequateliver-specific detoxification functionality. Hence in a preferredembodiment of the present invention a cell culture as defined hereinbefore is provided, characterized by transcript levels of CYP3A4 of atleast 70% of that of the in vivo level. More preferably, said transcriptlevel is at least 20, 40 or 100% of that of healthy adult liver.

In a particularly preferred embodiment, said differentiated cells havingconstitutively liver-specific metabolic activity are characterized by atleast two, preferably three of the following metabolic parameters:

a CPS expression level exceeding 20%, preferably exceeding 40%;a GS expression level exceeding 250%, preferably exceeding 300%; andan OTC expression level exceeding 25%, preferably exceeding 35%;

In another particularly preferred embodiment, said differentiated cellshaving constitutively liver-specific metabolic activity arecharacterized by at least four, more preferably all of the followingparameters:

a HNF4 expression level exceeding 250%, preferably exceeding 300%;a PXR expression level exceeding 70%, preferably exceeding 100%;a CPS expression level exceeding 20%, preferably exceeding 40%;an Arg I expression level exceeding 15%, preferably exceeding 35%;a GS expression level exceeding 250%, preferably exceeding 300%; anda CYP3A4 expression level exceeding 70%, preferably exceeding 100%;

As will be understood by the skilled person, each of the above recitedparameters define a functional capacity of a differentiated hepatocytecell line culture, i.e. an activity of which the differentiated cells ofsuch culture are capable. It will be understood that whether or not suchactivity is actually taking place will depend on external factors suchas the presence of the required substrates, ligands, targets, etc. inthe culture medium. Hence, whenever reference is made herein above to‘suitable culture conditions’, it will be clear to the skilled personthat, in essence, standard culture conditions are referred to, i.e.conditions under which cells can be kept while maintaining theirviability, with the additional requirement that such substrates,ligands, targets, etc. required for the respective activity are presentin the medium within given standard concentration ranges. It is in anycase within the skills of a trained professional to asses the capacityof a given human hepatocyte cell line culture to perform any of theaforementioned hepatic functions.

Method of Producing a Differentiated Hepatocyte Cell Culture

A second aspect of the present invention concerns a method of producinga cell culture comprising differentiated cells from a human hepatocytecell line in a medium, wherein said differentiated cells haveconstitutively liver-specific activity.

Said method comprises selection of a suitable cell line. A suitable cellculture may have low hepatic functions when the cells are in expansionphase, however at confluence these cells typically display transcriptlevels of transcription regulators HNF4 and PXR, of at least 20% of thelevels in vivo. More preferably, said transcript level is at least 30,50 70% and most preferably at least 75% of that of adult liver.

As noted above, a particularly suitable cell line for the purpose of thepresent invention is the cell line designated HepaRG or a cell line thatis clonally derived therefrom. Other cell lines that may suitably beused in accordance with this aspect of the invention include the celllines designated HepG2 and cBAL111 or cell lines clonally derivedtherefrom.

The present method includes expansion or proliferation of cells. Inpreferred embodiment of the invention, cells are expanded by culturing,typically in monolayer or in suspension, preferably attached to aculture surface, in a suitable medium, preferably comprising serum,hormones, growth-factors and anti-biotics. More preferably the medium atleast comprises serum, typically 2-10%, insulin, typically 2.5-10 μg/m1;cortico-steroid, typically 25-100 μM; penicillin and/or streptomycin,typically 0.5-2 U/ml; and glutamine, typically 1-5 mM. In a particularlypreferred embodiment a medium is used comprising Williams' E medium withapproximately 10% fetal bovine serum; approximately 5 μg/ml insulin,approximately 50 μM hydrocortisone, approximately 1 U/mlpenicillin/streptomycin and approximately 2 mM glutamine. After aproliferation phase, typically at confluence, the cells can be inducedto undergo differentiation. Alternatively, cells may be harvested and beused for further expansion.

In a preferred embodiment of the invention the cells are subsequentlycultured under conditions suitable for inducing differentiation. Forthis purpose the cells are typically cultured in a suitable mediumincorporating an effective amount of carbamoyl glutamate

(CG). The present inventors found that the presence of CG induces orenhances urea production capacity in the cells of the invention. Inaddition, the urea production from exogenously added ammonia istypically significantly enhanced. It was found, unexpectedly, thattreatment with CG has a preconditioning effect; when the cells areexposed to carbamoyl glutamate for at least 5 days, the increased ureaproduction will be maintained for at least 24 hrs upon withdrawal of theCG. Typically, the, instantaneous effect of CG is negligible. The cellculture medium thus preferably comprises an amount of CG of at least0.1, 0.2, or 0.5 mM, for example 1.0 mM. Furthermore, the culture mediummay typically comprise serum, hormones, growth-factors and antibiotics.More preferably the medium at least comprises serum, typically 2-10%,insulin, typically 2.5-10 μg/ml; cortico-steroid, typically 25-100 μM;

antibiotic, typically 0.5-2 U/ml; and glutamine, typically 1-5 mM. In aparticularly preferred embodiment a medium is used comprising Williams'E medium with approximately 10% fetal bovine serum, approximately 5μg/ml insulin, approximately 50 μM hydrocortisone hemisuccinate,approximately 1 U/ml penicillin/streptomycin and approximately 2 mMglutamine.

In a particularly preferred embodiment of the invention, the phase ofcell differentiation comprises culturing the cells in a culture mediumthat is substantially or completely free of DMSO, i.e. it preferablycontains less than 1%, 0.5%, 0.1%, 0.05%, 0.01% or 0.001% DMSO or itcontains 0% DMSO.

Furthermore, it is preferred that the complete culture medium employedfor the proliferation stage is also substantially or completely free ofDMSO. The inventors found that presence of DMSO drastically reduced thecell number, as deduced from the total protein content and inhibitedgene expression of synthetic and metabolic genes, as CPS and albumin andgalactose elimination.

During culturing, the cells are kept at a desired, biologically orphysiologically acceptable temperature, typically a temperature ofapproximately 37 ° C.

Preferably, during culturing, the cells are oxygenated using anoxygen-containing gas or gas mixture, such as pure oxygen, air, or a gasmixture containing oxygen, preferably 20-99% oxygen, typically about 40%oxygen, in admixture with another inert and/or physiologicallyacceptable gas such as nitrogen and carbon dioxide, typicallyapproximately 5% carbon dioxide. Furthermore, it is preferred that theatmosphere under which the cells are cultures is humidified.

Hence, in a particularly preferred embodiment of the present invention,a process is provided comprising:

selecting a human hepatocyte cell linea phase of cell proliferation comprising culturing the cells of saidhuman hepatocyte cell line in a suitable culture medium, preferablycomprising serum, hormones, growth-factors and antibiotics;a phase of cell differentiation comprising culturing the cells in asuitable culture medium comprising an effective amount of CG, saidculture medium furthermore preferably comprising serum, hormones,growth-factors and antibiotics.

In a preferred embodiment of the invention the cells are grown in athree-dimensional (3D) culture, typically on a three-dimensional supportmatrix material, as will be explained in more detail hereafter. Asindicated before, the present inventors have found that growing thecells in a 3D culture enhances the development of broad-spectrum hepaticfunctionality. In a preferred embodiment of the invention a process asdefined in any of the foregoing is provided, wherein the cells are grownand/or differentiated in a 3D culture, preferably on a three-dimensionalsupport matrix, e.g. in a bioreactor as described in more detailhereafter.

In a particularly preferred embodiment of the invention, a cell cultureis provided, obtainable by the above described processes.

In yet another embodiment, the invention provides a method of improvingthe functionality of hepatic cell line derived cultures by culturingthem in a culture medium containing CG and/or in the absence of DMSOand/or under conditions allowing for three-dimensional growth, inaccordance with what has been described in the foregoing.

In still another embodiment of the invention, the use of CG forimproving the functionality of hepatic cell line derived cultures isprovided, in accordance with what has been described in the foregoing.

Bioreactor

In a further aspect, the invention relates to a bioreactor comprising acell culture of the invention or obtained in a method of the inventionas described herein. The bioreactor of the invention may in principle beany suitable device for culturing and/or maintaining cells.

As stated in the introductory part of this application, many bioreactorconfigurations have been proposed and described. In this regardreference is made to Park and Lee' and Sgroi et al.² , the entirecontents of which are incorporated herein by reference, in particularthe subject-matter described therein, relating to bioreactorconfigurations.

As will be understood by the skilled person, a bioreactor will typicallycomprise a vessel or chamber, comprising a wall defining and enclosing athree-dimensional space, said vessel or chamber being suitable forholding solid and/or liquid content, e.g. a cell culture and a culturemedium. In accordance with the present invention the chamber or vesselis typically a closed system which does not allow for the exchange ofmatter between the space within the walls and the atmosphere surroundingsaid chamber or vessel. The wall of the reactor is thus typically bemade of any suitable inert and liquid and gas impervious material, suchas glass, plastic, e.g. plexiglass, polycarbonate or polysulfone, ormetal. From a practical viewpoint a material selected from the group ofpolysulfone is preferred, these materials being able to withstand(steam) sterilization conditions. The inside of the reactor vessel canbe provided with a special coating compatible with the cells to becultured, such as collagen or Matrigel.

The size of the reactor is not particularly limited and will usuallydepend upon the capacity required. The volume of the reactor cantherefore typically vary from 1 ml to 1000 litres. With a view to theintended incorporation of the bioreactor in a functional BAL system, asdescribed herein below, suitable reactor volumes are within the range of0.2-3 litre, most preferably within the range of 0.5-1 litre.

Typically, the bioreactor comprises a support matrix for attachment ofthe cells. The support matrix may e.g. comprise a three- dimensionalsolid support which may be in the form of a highly porous sheet or mat.Preferably such a three dimensional matrix comprises a physiologicallyacceptable network of fibers or a physiologically acceptable open-porefoam structure. Alternatively, the support matrix may be a semi-solidmaterial, such as a gel. Suitable examples of matrix materials includeGelfoam, PVF (Collagen coated Reticulated Polyvinyl formal resin),PVLA-RPU (Poly-N-para-vinylbenzyl-lactonamide coated reticulatedpolyurethane) PGA (Polyglycolic acid), PVA (Polyvinylalcohol), PGA/PLA(polyglycolic acid/polylactic acid), 3D-Polyurethane foam or non-wovenmatrix, and Porous siliconrubber foam. In a particularly preferredembodiment the support matrix has the form of rolled up or folded sheetsor mats. Although the solid support of the invention generally does notrequire a pre-treatment step before use, it is comprised within thescope of the invention to treat the support matrix with extracellularmatrix materials, such as Matrigel, poly-N-paravinylbenzyl-lactonamide,collagen based materials, or with gaseous compounds, in a manner knownper se, in order to further improve cell adhesion.

In a preferred embodiment of the invention the bioreactor comprisesmeans for supplying gaseous oxygen and for removal of gaseous carbondioxide. Such means may e.g. be in the form of conduits that arepermeable to gasses like oxygen and carbon dioxide. Preferably theconduits are evenly distributed throughout the three dimensional matrixand more preferably the distance between individual conduits is between0.05 mm and 5 mm Suitable conduits are e.g. hollow fibers or capillariesmade of a hydrophobic material and having an outer diameter of 0.1 mm to1.0 mm Preferably the bioreactor is provided with at least one gas inletand at least one gas outlet operably connected to said capillaries.

Furthermore, it is preferred that the bioreactor of the inventioncomprises means for perfusing the matrix with a liquid medium; typicallyit comprises at least one liquid inlet and one outlet, operablyconnected to the space containing the cells of the invention, throughwhich a liquid medium can be fed to this space and be brought intocontact with the cells and respectively be removed there from. Theseliquid in- and outlets may thus be used for perfusing a liquid mediumthrough the bioreactor whereby the cells of the invention in thebioreactor preferably produce, bioconvert or remove a substance in orfrom the medium.

Thus, typically, any bioreactor in accordance with this invention willat least comprise a chamber or vessel holding a matrix loaded with acell culture as described herein before, said bioreactor furthercomprising means for oxygenating said matrix.

The reactor can further comprise all known elements of biologicalreactors, such as gas and/or liquid pumps operably connected to thedifferent inlets or outlets; means for measuring and/or controlling thetemperature within the reactor vessel; access means, such as a hatch,for accessing the inside of the reactor; inspection means; means forseeding cells, such as additional inlets, means for intervening samplingof cells, probes and means for inserting them, such as probes for themeasurements of the viability as further described herein below, etc.The reactor may further be provided with means for the automatic controlof the different reactor functions, such as a computer means operablyconnected with the pumps, temperature controlling means etc.

The reactor may also be provided with means for agitating the reactor,such as an electric motor, for instance for rotating the reactor alongone of its axes, or with means for stirring inside the reactor, althoughthe latter is usually not preferred.

Method of Producing a Differentiated Hepatocyte Cell Culture in aBioreactor

A further aspect of the invention concerns the preparation of abioreactor comprising a differentiated hepatocyte cell culture.

In accordance with the invention, the cells are first expanded at largescale outside the bioreactor. This may be carried out by culturing thecells in suspension or, preferably, attached to a culture surface, whichmay be provided by e.g. roller bottles, multilayer culture plates orusing carriers in suspension in stirred or continuous-flow bioreactorsunder optimal oxygenation conditions. For harvesting, the cells will beexposed to a cell detachment solution of proteolytic and collagenolyticand DNAse enzymes. Preferably the cells are harvested with a 4:1 mixtureof Accutase™ and Accumax™ (Innovative Cell Technologies, Inc., SanDiego, USA).

The cells are introduced into a bioreactor as described herein before,after which they are allowed to attach and/or adhere to the supportmatrix during a suitable period of time. In cultivating the cells, thereactor can be loaded with a small amount of cells, after which thecells are allowed to divide so as to fully populate the reactor.According to this embodiment, the support matrix will only requireloading of amounts of as low as 5% of the total cell capacity, so as tofully populate the reactor by advantageous “three dimensional” growth.Preferably, the reactor is loaded with an amount of 10-80%, morepreferably 40-80%, more preferably 50-80%, most preferably 60-80%, ofthe total cell capacity. It is also possible to feed more cells into thereactor, so as to fully saturate the matrix material with adherentcells, or even to use an excess amount of cells, after which superfluouscells are removed. In general, the BAL will be seeded with 10⁵-10⁹cells/ml, usually around 10-500*10⁶ cells/ml and/or with an amount ofcells corresponding to at least 5%, 10%, 15% or 20% of normal livermass. The reactor is usually seeded by injecting a suspension of thecells into the reactor, after which the cells are allowed to distributethemselves throughout the reactor and adhere themselves to the solidsupport during a suitable period of time. In order to facilitate thedistribution of the cell suspension even further, the reactor can beagitated after the cell suspension has been injected. According to ahighly preferred embodiment, after injection of the cell suspension, thereactor is rotated, preventing the formation of a cell pellet at thebottom in the bioreactor, typically for 2-4 hours. After immobilisationof the cells is complete, the remaining suspension containingnon-adhered and/or excess cells is removed from the reactor, by flushingthe reactor with a suitable liquid medium.

After loading of the cells and adhering of cells to the matrix, thebioreactor is perfused with culture medium as described herein before,typically at a rate of 50-250 mL/min , e.g. at 150 mL/min, typicallysuch as to allow them to undergo one or several population doublings. toFurthermore, this process typically allows for the cells to reorganizein 3D, a process which typically enhances the hepatic functionality ofthe cells eventually obtained, as indicated herein before. The processis preferably continued until the cell culture in the bioreactor stopsproliferating. This can be calculated from the loading mass as deducedfrom cell number, total protein or DNA content and previously determinedpopulation doubling time of the cell line in a bioreactor. It can alsobe deduced from the stabilization of oxygen consumption.

Subsequently, the reactor is perfused with culture medium to inducedifferentiation. During the differentiation phase, culturing hepatocytecell lines, in particular the HepaRG cell line, in the bioreactor canupregulate their hepatic functions to an unexpectedly high degree. Theinventors found a significant improvement of urea production and ammoniaelimination and conversion of lactate production into lactateconsumption and a significant increase of expression levels of CYP2B6and CYP3A4 when HepaRG cells were cultured in a bioreactor under theconditions as described herein. Hence, in a particularly preferredembodiment of the invention, a method of producing a bioreactor isprovided, comprising:

a phase of cell proliferation comprising culturing cells of a humanhepatocyte cell line in a suitable culture medium, preferably comprisingserum, hormones, growth-factors and antibiotics;loading of viable cells to an amount of at least 5% of normal liver massin a bioreactor comprising a support matrix, and allowing the cells toattach to said matrix;an expansion or proliferation phase comprising culturing the cells in asuitable culture medium, preferably comprising serum, hormones,growth-factors and antibiotics, to achieve at least one populationdoubling; anda differentiation phase comprising culturing the cells in a culturemedium comprising serum, hormones, growth-factors and antibiotics.

In a preferred embodiment of the invention, during the differentiationphase, said culture medium comprises a suitable amount of CG, e.g. anamount of at least 0.5 mM, such as to further enhance urea productioncapacity of the cells, as explained herein before. In a particularlypreferred embodiment of the invention, the culture media employed insaid proliferation and differentiation phases are substantially orcompletely free of DMSO, as explained herein before.

The differentiation phase typically includes at least 7 days, morepreferably at least 14, 21 or 28 days and up to 50 days.

Hence, in a particularly preferred embodiment of the invention, a methodof producing a differentiated hepatocyte cell culture in a bioreactor isprovided, comprising:

selecting a human hepatocyte cell linea phase of cell proliferation comprising culturing cells of a humanhepatocyte cell line in monolayer or in suspension, preferably attachedto a culture surface, in a suitable culture medium, preferablycomprising serum, hormones, growth-factors and antibiotics;loading of viable cells to an amount of at least 5%, preferably 40-80%,of normal liver mass in a bioreactor comprising a support matrix, andallowing the cells to attach to said matrix;a potential expansion or proliferation phase and reorganization in 3Dphase comprising culturing the cells in a suitable culture medium,preferably comprising serum, hormones, growth-factors and antibiotics;anda differentiation phase of at least 7 days comprising culturing thecells in a culture medium comprising an effective amount of CG, saidmedium further preferably comprising serum, hormones, growth-factors andantibiotics.

During culturing, the cells are kept at a desired, biologically orphysiologically acceptable temperature, typically a temperature ofapproximately 37° C., e.g. by keeping the reactor in a thermostat, or bycontrolling the temperature of liquid and/or gas flows, as will be clearto one skilled in the art. The gas and liquid are preferably leadthrough the bioreactor continuously at suitable flow rates, using theafore mentioned liquid and gas in- and outlets of the reactor.

BAL System

A further aspect of the invention concerns a BAL system comprising abioreactor as described herein before. The advantageous properties ofthe cells and the bioreactor of the invention make them especiallysuited for use in or as a bio-artificial liver system.

Therefore, in general, the bio-artificial liver system of the inventioncomprises a bioreactor of the invention comprising a support matrixloaded with a cell culture as defined hereinabove.

During use of the present BAL system, the bioreactor liquid medium in-and outlets are operably connected to the blood circulation of a subjectby means of a liquid circuit, so that a liquid medium directly orindirectly derived from the subject is used to perfuse the bioreactor,allowing said cells to carry out functions normally carried out by theliver in vivo. Liquid medium leaving the bioreactor is returned to thesubject directly or indirectly. The BAL-system of the invention willtherefore comprise a liquid circuit for circulating the liquid tomedium, as well as one or more pumps for controlling the liquid flowthrough said circuit.

The circuit can also contain further appliances for the (pre-)treatmentof the liquid medium, i.e. blood or plasma, such as an activatedcharcoal column for the absorption of hydrophilic toxins and/or a resincolumn for adsorption of hydrophobic substances (e.g. bilirubine). Othertypes of such detoxification systems or columns known to the skilledperson may be included in the system as well or instead, withoutdeparting from the scope of the invention.

The liquid circuit may also comprise means for adding nutrients andother desired substances to the liquid medium, although in this respectthe liquid medium derived from the patient may itself be sufficient forkeeping the liver cells in the reactor viable. The same applies withregard to oxygen requirements of the cells; separate supply of oxygendoes not need to be provided during perfusion of the bioreactor with anoxygen containing medium, such as blood. Preferably however the systemdoes include a source of oxygen or oxygen containing gas. Such anoxygenation system is operably connected to a bioreactor gas inlet and asystem for removing gas is operably connected to the bioreactor gasoutlet. As will be understood by the skilled person, a pump may beconnected to any gas conduit connected to the bioreactor gas in- andoutlets.

During use, the Bioreactor of the invention can be perfused with wholeblood—either arterial or venous—derived from a subject in a manner knownper se, but preferably the bioreactor is perfused with plasma obtainedfrom the blood. In this preferred embodiment, the BAL-system's liquidcircuit will usually comprise a plasma separator or plasmapheresis unitfor separating the plasma from the whole blood derived from the subject.The use of BAL-systems on the basis of plasmapheresis, as well assuitable plasmapheresis units, are well known in the field and are forinstance described in V.d. Kerkhove et al.¹³, which is to be consideredincorporated herein by reference.

The liquid circuit will also typically contain one or more cellfiltering units, such as membrane filters, columns or hollow fibremodules with a suitable molecular weight cut off, either placed beforeor after the reactor. Since the present invention may employ tumorigeniccells, it is particularly preferred that one or more filtering units areincorporated in the circuit, placed downstream of the bioreactor, i.e.at the bioreactor liquid outlet side, with a view to reducingtumorigenicity associated risks for the subject to be treated.

Although dependant upon the geometry and capacity, the amount andactivity of the cells present in the reactor, the desired therapeuticapplication and other such factors, the BAL-system of the invention istypically suitable for treating 1 to 300 ml of liquid medium derivedfrom a patient per minute. In order to achieve this, the liquid mediumcan be fed directly to the reactor at a corresponding rate, e.g. of 50mL/min Preferably, the bioreactor of the invention is incorporated intoa “high flow loop”, as known per se from the abovementioned prior art.In such a loop, the flow of the liquid medium over the reactor can bekept at a higher or lower rate than the flow of liquid from the patientthereby providing for recirculation of the liquid medium over thereactor. Usually, this will be carried out by suitable control of thedifferent pumps in such a high flow loop system, by keeping them at asuitable flow ratio.

The BAL system preferably comprises a single bioreactor as defined abovecomprising a cell culture in accordance with the invention. However, aBAL system may comprise a bioreactor connected in series and/or inparallel with one of more other bioreactors, typically containingdistinct cell cultures or an artificial detoxification system. Hence, aBioartificial liver device is also provided comprising a bioreactor asdefined above comprising a cell culture in accordance with the inventionin combination with another bioreactor comprising a chamber or vesselholding a matrix loaded with a cell culture containing differentiatedcells of a human cell line, and furthermore comprising means foroxygenating said matrix, wherein the cells at least differ in theirphenotype compared to the first bioreactor. Alternatively, thebioreactor as defined above comprising a cell culture in accordance withthe invention is combined with an artificial detoxification module, e.g.a charcoal column or an albumin dialysis system.

In accordance with the present invention, it is preferred to combine inone BAL system modules providing distinct functionalities if a singlebioreactor does not fully cover the functional spectrum or is tosusceptible to detrimental effects of liver failure plasma or blood ofthe treated subject. More specifically, an artificial detoxificationmodule or one or more further bioreactors may be combined with theliver-specific bioreactor of the present invention, said furtherbioreactors providing e.g. supplementary liver-specific detoxificationfunctionality and/or immunologic functionality. Such a composite BALcomprises at least two consecutive bioreactors with cells differentiatedinto diverging directions: the first with detoxification phenotypepreceding the second liver-specific bioreactor of the present invention.Alternatively, the composite BAL comprises an artificial detoxificationmodule preceding the liver-specific bioreactor of the present invention.The plasma of the patient will be perfused through the detoxificationbioreactor or module, to specifically eliminate and inactivate compoundstoxic to the patient, which may also be toxic to the cells loaded in thebioreactor of the present invention. Subsequently the plasma will beperfused through the bioreactor of the present invention which willexecute most liver-specific functions, like ammonia elimination, ureaproduction and synthesis of plasma proteins etc. A bioreactor withimmunomodulatory characteristics can optionally be included after thebioreactor of the present invention.

The different elements of the BAL-circuit may be provided as anintegrated system in a single housing, or the BAL may consist ofseparate connected elements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates a high flow loop two-reactorbioartificial liver system including a plasmaphoresis unit according toan embodiment of the invention.

FIG. 2 schematically illustrates a one-reactor bioartificial liversystem including a plasmaphoresis unit according to an embodiment of theinvention.

A possible configuration of a particularly preferred high flow looptwo-reactor BAL system comprising a plasmapheresis unit is shownschematically in FIG. 1. Said figure shows a subject (1) to be treated,connected to the BAL system (2), which comprises a blood circuit (3) anda plasma circuit (4). The blood circuit comprises a plasmapheresissystem (5), that separates plasma from the subject's blood, which plasmais lead to a plasma reservoir (6). Blood flowing out of theplasmapheresis system is combined with plasma coming from the plasmareservoir (6) and lead back to the subject. The blood circuitfurthermore comprises a pump (8), set to produce a flow of blood of e.g.approximately 50 ml/min Additional pumps (9, 10), a heater (11) and afilter (12) are incorporated in the plasmapheresis system - plasmareservoir loop (7). The plasma circuit comprises a detoxificationbioreactor (13) and a metabolic bioreactor (14) connected in series. Theplasma reservoir (6) is operably connected to the detoxificationbioreactor; the detoxification reactor is operably connected to themetabolic bioreactor; and the metabolic bioreactor is operably connectedto the plasma reservoir. Each reactor comprises a gas inlet operablyconnected to a source of a gas, such as a gas cylinder (21, 22) and agas outlet system (23, 24). The bioreactor loop furthermore includes aheater (25), a pump (26), set to produce a flow of plasma of e.g.approximately 150 ml/min , and a filter (27).

A possible configuration of a particularly preferred one-reactor BALsystem comprising a plasmapheresis unit is shown schematically in FIG.2. Said figure shows a subject (1) to be treated, connected to the BALsystem (2), which comprises a blood circuit (3) and a plasma circuit(4). The blood circuit comprises a plasmapheresis system (5), thatseparates plasma from the subject's blood, which plasma is lead to aplasma reservoir (6). Blood flowing out of the plasmapheresis system iscombined with plasma coming from the plasma reservoir (6) and lead backto the subject. The blood circuit furthermore comprises a pump (8), setto produce a flow of blood of e.g. approximately 50 ml/min Additionalpumps (9, 10), a heater (11) and a filter (12) are incorporated in theplasmapheresis system-plasma reservoir loop (7). The plasma reservoir(6) is operably connected to the bioreactor (28) via the plasma circuit.The bioreactor comprises a gas inlet operably connected to a source of agas, such as a gas cylinder (21) and a gas outlet system (24). Thebioreactor loop furthermore includes a heater (25), a pump (26), set toproduce a specific flow of plasma and a filter (27).

Treatment of Hepatic Failure

The cell cultures of the invention as well as the bioreactors andbioartificial liver systems containing them can be used to supportand/or replace liver function in subjects with impaired liver functionor in other cases in which artificial liver support is desirable and/orrequired. Hence further aspects of the invention concerns use of thecell cultures, bioreactors and/or BAL systems as described herein beforefor treating a subject in need of such treatment, typically fortemporarily replacing and/or supporting hepatic functions in a subjectin need thereof, as well as methods of treatment of such subjects, saidmethod comprising extra-corporeal or ‘liver support’ employing said cellcultures, bioreactors and/or BAL-systems of the invention.

Typically, these aspects of the invention concern treatment of subjectssuffering from acute liver failure, end stage liver disease oracute-on-chronic liver disease. Examples of these conditions may includefulminant hepatic failure (FHF), for instance due to viral hepatitisinfections or acute liver poisoning (for instance with acetaminophen,CC14, alcohol or drugs), as well as transient liver ischaemia, and livertrauma due to injury. It may also concern treatment to improve thesubjects condition before liver transplant, to bridge the period beforeliver transplant, to bridge the rejection period after acute rejectionof a transplanted liver, during the anhepatic phase while a livertransplant is carried out and/or during recovery of a liver transplant,or to allow time to regenerate the patient's own liver. Furthermore, itmay concern treatment of subjects suffering from chronic liver diseasesto enhance the quality of life of the patient and/or to bridge periodsof exacerbation.

Treatment of the subject will typically involve perfusing the cellcultures contained in the BAL-system bioreactors with the subject'sblood or plasma. Preferably this is done continuously for a period oftime sufficient to stabilise and preferably improve haemodynamic andbiochemical parameters, typically meaning that an amount of cells withinthe range of 5%, 10%, 20% or 30% of liver mass is perfused with thesubjects plasma for 8-24 hours at a flow rate of 20-70 mL/min.

Before or during use, the functional effect and the metabolicperformance of the bioreactor and the cells contained therein can bemonitored in a manner known per se with any of the large number of testsavailable for this purpose, such as measurement of protein synthesis,ureagenesis, oxygen uptake (for which advantageously direct measurementat the gas inlet and gas outlet can be used), cytochrome P450-activity,drug metabolic assays, clearance techniques etc, see for instance Rozgaet al.¹⁴, incorporated herein by reference. Also, a biomass meter can beused, which uses conductivity measurements based upon differences inmembrane potential between dead cells and living cells. Such a meter isknown to one skilled in the art.

An advantageous aspect of the present invention resides in thepreconditioning of the cell cultures using CG during culturing of thecells, such that the cells need not be exposed to (additional) CG duringtreatment of the patient in order to retain sufficient hepaticfunctionality for a suitable period of time. Hence in a preferredembodiment of the invention a method of treatment of a subject isprovided, as defined herein before, wherein no external source of CG isadded to the cell cultures during use of the bioreactor.

The different aspects of the invention as described here above will beillustrated by means of the following non-limiting examples.

EXAMPLE 1 Preparation of HepaRG Cell Cultures in Monolayer HepaRGCulture in Monolayer

HepaRG cells are primarily expanded in monolayer at 37° C. in ahumidified atmosphere (95% air, 5% CO₂), since proliferation reduceswhen the cells are cultured in the bioreactor. The culture medium(HepaRG medium) consists out of Williams' E medium (Lonza) with 10%fetal bovine serum (FBS, Lonza), 5 μg/ml insulin (Sigma), 50 μM tohydrocortisone hemisuccinate (Sigma), 1 U/ml penicillin/streptomycin(Lonza) and 2 mM glutamine (Lonza). The applied FBS batch should betested before large expansion of the cells will be carried out (seebelow).

Testing FBS Batch and Functionality in Monolayer

The performance of the HepaRG cells can be tested in monolayer tooptimize culture conditions that may be applicable to BAL cultures aswell. Furthermore the appropriate FBS batch can be selected, this way.Differences in functionality of the cells were found, depending on thebatch of FBS used. Therefore the batch should be tested thoroughlybefore large scale culturing in Hyperflasks™ (Coming) and the bioreactorwill be pursued.

For testing, the cells are seeded in 6-well and 24-well culture plates(Corning) in HepaRG media with different FBS batches or otherdifferences in medium composition. After a 2 weeks proliferation phase,2% of DMSO will be added to half of the cultures. After another 2 weeksof culturing, the confluence and morphology of the cultures will beassessed microscopically. Furthermore the +DMSO and −DMSO cell culturesin the 24-well plates will be tested for biochemical activity, i.e. ureaproduction, ammonia elimination and protein content. The cultured cellsare washed twice using phosphate buffered saline (PBS, NBPIInternational) and then incubated with test medium consisting of HepaRGculture medium supplemented with 1.5 mM ¹⁵NH₄Cl (Sigma), 2.75 mMD-galactose (Sigma), 2 mM ornithine hydrochloride (Sigma) and 2 mML-lactate (Sigma). Medium samples are taken after 1 hr from the culturewells, as a reference, and after 24 hours. At the end of the test periodthe cells are washed three times using PBS and stored at −20° C. forprotein determination. From the changes in concentration at t=1 hr and24 hr of urea and ammonia an activity per hour and per mg protein/wellis calculated. Quality of the cell-loaded bioreactors, measured byhepatocyte function at the start of the treatment, will be compared withvalues of primary hepatocytes that have been tested extensively in vitroin the past.

The HepaRG cells cultured in the 6-well plates are lysed and the RNA isisolated using the RNeasy kit (Qiagen). Next, real-time reversetranscriptase-polymerase chain reactions (RT-PCR) are performed toquantify mRNA levels of hepatic genes as well as 18S ribosomal RNAlevels by RT-PCR.

Biochemical Analysis

Ammonia: Ammonia concentrations are determined by an enzymatic kineticcolorimetric assay using glutamate dehydrogenase and NADPH, using theAmmonia (rapid) kit (Megazyme, Ireland) according to the instructions ofthe manufacturer.

Testosterone: Testosterone concentrations are determined highperformance liquid chromatography coupled to mass spectrometry.

Urea: Urea concentrations of test medium samples are determined usingthe blood urea nitrogen test of Sigma Chemical Co according to theinstructions of the manufacturer.

¹⁵N Urea: Single and Double labelled urea is measured in the test mediumsamples using mass-spectrometry.

Albumin: Human albumin concentrations are determined by ELISA usinggoat-anti-human serum albumin antibody (1:100, Abcam, ab8940;) and HRPconjugated rabbit-anti-human serum albumin (1:5000, Abcam, ab7394).

ApoA1: Human ApoA1 concentrations are determined by ELISA using rabbitanti-human ApoA1 (1:500, Calbiochem, 178422), as first antibody,monoclonal mouse anti human ApoA1 (1:500, Calbiochem, 178472) as secondantibody and polyclonal rabbit anti mouse IgG-HRP (1:2000, Dako Po 260)as third antibody.

AST and LDH: Both AST and LDH activities are analyzedspectrophotometrically using a P800 Roche Diagnostics analyzer.

Lactate and glucose concentration: Both are analyzed using a P800analyzer (Roche Diagnostics).

Total protein: Total protein was quantified by spectrometry usingCoomassie blue (Bio-Rad).

RT-PCR

First-strand cDNA is generated using a combination of 40 pmol ofgene-specific RT-primers (see Table 1) and 134 units of Superscript III(Invitrogen) and 1 μg RNA in a total volume of 25 μl at 50° C. for onehour followed by a 15 min incubation at 70° C. The resultinggene-specific RT reaction mixtures are column purified using QiaquickPCR Purification Kit (Qiagen).

Real-time PCR is performed using 1 μl of cDNA and LightCycler FastStartDNA Master SYBR® Green 1 reagent (Roche). PCR primers are depicted inthe Table 3. The thermal cycling profile of the touchdown PCR is asfollows: 94° C. for 10 min, followed by 40 cycles of 94° C. for 1 s,primer annealing at 68° C. for 7 s with a 0.5° C./cycle decrease until63° C., and extension for 40 s at 72° C. PCR specificity is verified bymelting curve analysis and agarose gel electrophoresis. The mRNAquantification is carried out by using the LinRegPCR programme (Ramakerset al.¹⁷). For each sample the mRNA starting level is normalised for the18S rRNA starting level. To correct for variations between RT-PCR runs,the mRNA starting levels of liver specific genes of NKNT-3 cells areadditionally standardized for average mRNA starting levels of two liversamples that are simultaneously subjected to RT-PCR.

TABLE 1 Primer sequences Gene Primer 5′-3′ RT GCATCGCCGGTCGGCATCG (SEQID NO: 1) 18S S TTCGGAACTGAGGCCATGAT (SEQ ID NO: 2) rRNA ASCGAACCTCCGACTTTCGTTCT (SEQ ID NO: 3) RT GGGGGATAGACATGGGTATGG (SEQ IDNO: 4) AAT S ACAGAAGGTCTGCCAGCTTC (SEQ ID NO: 5) AS GATGGTCAGCACAGCCTTAT(SEQ ID NO: 6) RT CGTTTTGTCTTCTCTTCCCC (SEQ ID NO: 7) AFP STKCCAACAGGAGGCYATGC (SEQ ID NO: 8) AS CCCAAAGCAKCACGAGTTTT (SEQ ID NO:9) RT ACTTCCAGAGCTGAAAAGCATGGTC (SEQ ID NO: 10) ALB STGAGCAGCTTGGAGAGTACA (SEQ ID NO: 11) AS GTTCAGGACCACGGATAGAT (SEQ ID NO:12) RT TGTGATTACCCTCCCGAGCAAGTC (SEQ ID NO: 13) Arg I STTGGCAAGGTGATGGAAGAAACA (SEQ ID NO: 14) AS CCTCCCGAGCAAGTCCGAAACAA (SEQID NO: 15) RT ACAAGGGCAGAAAAGAAAAGGAGT (SEQ ID NO: 16) Arg II SGGTCCCGCTGCCATAAGAGA (SEQ ID NO: 17) AS GGCATCAACCCAGACAACACAA (SEQ IDNO: 18) RT CTGCAGTGACAGCTGGTTGAGG (SEQ ID NO: 19) ASL SCTGGAGCCACTGGATTCTGAG (SEQ ID NO: 20) AS GCCCCAAAGTTGAGTTCTGCT (SEQ IDNO: 21) RT CCTGAGGGAATTGATGTTGATGAA (SEQ ID NO: 22) ASS SCGTGGGCCGTATTGACATCGTG (SEQ ID NO: 23) AS CCGGTGGCATCAGTTGGCTCATA (SEQID NO: 24) RT AGGACCCGCACTGCTGGAGAAG (SEQ ID NO: 25) CPS I SCATCAGACTGGCTCAAAC (SEQ ID NO: 26) AS CAGCTGTCCTCCGAATCAC (SEQ ID NO:27) RT TCAGGTCGACTTTCACGCCC (SEQ ID NO: 28) CYP1A2 SGGAGGCCTTCATCCTGGAGA (SEQ ID NO: 29) AS TCTCCCACTTGGCCAGGACT (SEQ ID NO:30) RT GTTGGCGGTAATGGACTGGAAGA (SEQ ID NO: 31) CYO2B6 SCCCGCCCTCTGCCCCTTTTG (SEQ ID NO: 32) AS TCCACACTCCGCTTTCCCATCC (SEQ IDNO: 33) RT CTCTTTCAGCCAGTGGGAAA (SEQ ID NO: 34) CYP2C9 STCCTTGTGCTCTGTCTCT (SEQ ID NO: 35) AS ATCCATGCAGCACCACTA (SEQ ID NO: 36)RT GCTTCACAAAGTGGCCCTGG (SEQ ID NO: 37) CYP2D6 S CCTGCGCATAGTGGTGGCTG(SEQ ID NO: 38) AS GCTTCTCCCAGACGGCCTCA (SEQ ID NO: 39) RTAAAGAATGGATCCAAAAAATCA (SEQ ID NO: 40) CYP3A4 S AGTGTGGGGCTTTTATGATGG(SEQ ID NO: 41) AS AAGGCCTCCGGTTTGTGAAG (SEQ ID NO: 42) RTGGTACCATCTCTTGAATCCACC (SEQ ID NO: 43) CYP3A5 S TGACCCAAAGTACTGGACAG(SEQ ID NO: 44) AS TGAAGAAGTCCTTGCGTGTC (SEQ ID NO: 45) RTAGCCAAATCTACTTCCCCAGCAC (SEQ ID NO: 46) CYP3A7 SATTACGCTTTGGAGGACTTCTTCT (SEQ ID NO: 47) AS CGTCTTCATTTCAGGGTTCTATTT(SEQ ID NO: 48) RT TTGGCAGAGGGGCGACGAT (SEQ ID NO: 49) GS SGCCTGCTTGTATGCTGGAGTC (SEQ ID NO: 50) AS GGCGCTACGATTGGCTACAC (SEQ IDNO: 51) RT AGCAGGTCCAGCAGGTTG (SEQ ID NO: 52) GSTπ S GCCAGAGCTGGAAGGAGG(SEQ ID NO: 53) AS TTCTGGGACAGCAGGGTC (SEQ ID NO: 54) RTCACTCCAACCCCGCCCCTC (SEQ ID NO: 55) HNF4 S TCCGGGCTGGCATGAAGAAGG (SEQ IDNO: 56) AS CCAGGGGGAGCTCGCAGAAAG (SEQ ID NO: 57) RT CAGCTGCTGGGAAATGGTG(SEQ ID NO: 58) HNF6 S CCGGCCGGGAGACCTTC (SEQ ID NO: 59) ASAGAGTTCGACGCTGGACATC (SEQ ID NO: 60) RT GACGATGATGGTGAAGACAGGAG (SEQ IDNO: 61) MRP2 S AGCACCGACTATCCAGCATCTC (SEQ ID NO: 62) ASATCCGGCCTGTGGGTGTTGTG (SEQ ID NO: 63) RT GTAGGTGCCATTTCCCAGAGC (SEQ IDNO: 64) NTCP S GGCTTTCTGCTGGGTTATGTT (SEQ ID NO: 65) ASGGGGAAAGAAGAAAAGTGGTC (SEQ ID NO: 66) RT ATCATCTCTTGGGCATTCACC (SEQ IDNO: 67) RT CATGTGGGGCAGCAGGGAGAAG (SEQ ID NO: 68) OTC SGCCGGATGCTAGTGTAACCAA (SEQ ID NO: 69) AS AGCCGCTTTTTCTTCTCCTCTTC (SEQ IDNO: 70) RT GGCAAATCCCACCAACTCCAC (SEQ ID NO: 71) PXR SCGCCTGCGCAAGTGCCTGGAG (SEQ ID NO: 72) AS GTCGGCTGGGGGTTTGTAGTTC (SEQ IDNO: 73) RT CCAGACCACACTTGCCCGCTATG (SEQ ID NO: 74) RT, reversetranscriptase primer; S, sense PCR primer; AS, anti-sense PCR primer.AAT, α-1-antitrypsin; AFP, α-fetoprotein; ALB, albumin; Arg, arginase;ASL, argininosuccinate lyase; ASS, argininosuccinate synthetase; CKcytokeratin; CPS, carbamoyl-phorsphate synthetase; Cyp, cytochrome P450;GS, glutamine synthetase; GST, glutathione S transferase; HNF, hepaticnuclear factor; MRP, multidrug resistance protein; NTCP, NA-dependentcholate transporting protein; OTC, ornithine transcarbamylase;; PXR,pregnane X receptor.

Characterization Human Hepatocyte Cell Lines in Monolayer

The HepaRG cells, cultured in monolayer in 24 well plates in presenceand absence of DMSO as indicated above, were assessed for hepaticfunctions. For comparison, functions of hepatocyte cell lines NKNT-3,HepG2 and cBAL111, cultured under their respective optimal conditions,described by Hoekstra et al ¹⁸, and Deurholt ¹⁹, are given as far asdetermined Results are shown in Table 2.

Table 2 shows that HepaRG cells outperformed the NKNT-3, HepG2 andcBAL111 cells in exhibiting ammonia elimination, a critical parameter inacute liver failure. Since NKNT-3 cells did not exhibit any positivemetabolic parameter, the investigation of these cells was not furtherpursued. Comparison of the expression profiles of the HepG2, cBAL111 andHepaRG cells showed that genes expressed in highly differentiatedhepatocytes, like CYPs, Arg1 and CPS were most consistently expressed inthe HepaRG cells. The HepaRG cells treated with DMSO showed highermetabolic parameters compared to untreated HepaRG cells when normalizedfor total protein content. However the DMSO treatment killed more than50% of the cells, as can be deduced from the total protein/well contentand also from the increased cell leakage. The absolute metabolicparameters were therefore higher when DMSO treatment was omitted in theHepaRG cells, see Table 3. Furthermore, DMSO treatment increased CYP2B6and 3A4 expression, but other hepatic genes tested were eitherunaffected or decreased in their expression (Table 2).

TABLE 2 Characteristics of hepatocyte cell lines in comparison withprimary human hepatocytes in monolayer. NKNT-3 (reverted) HepG2 cBAL111(d18) HepaRG + DMSO HepaRG − DMSO PHH total protein/well (μg) 200 260 ±8  183 ± 21 109 ± 21  247 ± 47  85.2 ± 30  Biochemical functions Ammoniaelimination (nmol/h/mg protein) production undetectable undetectable 107± 41  72 ± 21  95 ± 5.0 Urea production (nmol/h/mg protein) undetectable3.3 ± 3.7 8.0 ± 6.6 8.1 ± 6.8 5.0 ± 2.0 96 ± 34 Lactate elimination(μmol/h/mg protein) ND ND ND production production ND Albumin production(ng/h/mg protein) undetectable 2.8 ± 0.3 8.0 ± 6.6 ND ND 37.7 ± 7.7 ApoA1 production (μg/h/mg protein) ND ND ND 1.06 ± 0.95 0.78 ± 0.50 0.61± 0.16 6bOH testosteron production (nmol/h/mg ND 0.055 ± 0.006 ND ND ND61.7 ± 8.2  protein) mRNA levels (% of in vivo) PXR ND 20 ± 8  11.1 42 ±19 44 ± 16 ND HNF4 ND 484 ± 181 57.0 61 ± 21 88 ± 89 ND CYP2B6 ND <124.0 61 ± 30 3 ± 1 ND CYP3A4 ND <1 2.7 69 ± 47 10 ± 8  ND ALB 24 ± 22 63± 6  2.0 23 ± 4  66 ± 24 ND ARG1 ND 11 ± 5  1.7 5 ± 4 15 ± 11 ND CPS 1.3± 1.4 3 ± 1 5.5 1.1 ± 0.4 31 ± 19 ND GS ND 1564 ± 624  89.7 90 ± 29 214± 66  ND (ND = not determined) (CPS, carbamoyl phosphate synthetase;ArgI, arginase I, GS, glutamine synthetase; ArgII, Arginase II; HNF4,hepatocyte nuclear factor 4; PXR, pregnane X receptor; CYP2B6,cytochrome P450 2B6; CYP3A4, cytochromeP450 3A4; Alb, albumin)

TABLE 3 Characteristics of −DMSO and +DMSO cultures of HepaRG cells (n ≧10 from ≧3 independent experiments). Values are given as mean ± SD. Pvalues refer to −DMSO vs +DMSO cultures. Parameter −DMSO +DMSO P valueAbsolute Total protein (μg/well) 247 ± 47  109 ± 21  <0.001 valuesAmmonia elimination (nmol/h) 17.0 ± 4.6  11.6 ± 4.3  <0.001 Ureaproduction (nmol/h) 1.2 ± 0.5 0.8 ± 0.5 0.003 Galactose elimination(nmol/h) 26 ± 5.4 7.2 ± 1.8 <0.001 ApoA1 production (μg/h) 0.17 ± 0.110.11 ± 0.11 ns AST leakage (mU/h) 0.27 ± 0.07 0.43 ± 0.08 <0.001 LDHleakage (mU/h) 1.54 ± 0.24 1.87 ± 0.43 <0.001 Protein Ammoniaelimination (nmol/h/mg protein) 72 ± 21 107 ± 41  <0.001 normalized Ureaproduction (nmol/h/mg protein) 5.0 ± 2.0 8.1 ± 6.8 0.02 values Galactoseelimination (nmol/h/mg protein) 100 ± 24  58 ± 17 <0.001 ApoA1production ((μg/h/mg protein) 0.78 ± 0.50 1.06 ± 0.95 ns AST leakage(mU/h/mg protein) 1.3 ± 0.4 4.9 ± 2.4 <0.001 LDH leakage (mU/h/mgprotein) 7.4 ± 2.2 21.2 ± 9.4  <0.001 (ns = not significant)

Preconditioning Effect of Carbamoyl Glutamate and Conversion of Ammoniainto Urea

The HepaRG cells are cultured as indicated above in 24 well in presenceor absence of 1 mM CG during the last 5 days. Next the cells are testedas indicated above in presence or absence of CG during 24 hours. Todetermine the conversion of ammonia into urea, the ¹⁵N urea enrichment(single and double labeled) is measured in the test samples usingmass-spectrometry. Similarly, HepG2 and cBAL111 cells were tested fortheir response to CG. Results are shown in Table 4.

TABLE 4 Urea production of HepG2, cBAL111 and HepaRG cells, cultured inmonolayer. Carbamoyl glutamate (CG; 1 mM) addition was varied before andduring the incubation with test medium. (−−, no CG; −+, CG only duringtest; +−, CG only during preincubation; ++, CG in preincubation and intestmedium). Urea production is expressed as nmol/h/mg protein and 15Nurea enrichment is given as % of total urea produced. CG Urea production¹⁵N urea enrichment Treatment HepG2 cBAL111 HepaRG + DMSO HepaRG − DMSOHepaRG + DMSO HepaRG − DMSO −− 1.0 ± 1.7 undetectable  6.0 ± 4.2 4.1 ±2.2 15.5 ± 8.4  6.4 ± 4.7 −+ 1.8 ± 1.1 8.3 ± 8.3  4.9 ± 4.2 5.9 ± 2.4 NDND +− 2.5 ± 1.4 10.8 ± 6.2  11.0 ± 5.3 9.6 ± 3.3 ND ND ++ 2.4 ± 1.6 9.4± 8.4 13.7 ± 4.4 9.5 ± 4.2  7.8 ± 2.4 24.4 ± 4.1 (ND = not determined)

These results show that CG treatment increases urea production more thantwo-fold in all cell lines. In HepaRG-DMSO cells the % of urea of thetotal urea produced from ammonia increased even four-fold. Furthermorepre-treatment with CG is sufficient for increasing the urea production,since the ureagenesis is similar in the +− and ++ cultures. Thereforethe beneficial effects of CG on urea production are preserved even whenCG is omitted from the test or during a clinical treatment. No effect ofCG on other hepatic functions and the gene expression profiles werefound.

EXAMPLE 2 Preparation of a Bioreactor Comprising Differentiated HepaRGCell Cultures and Evaluation of their Metabolic and DetoxificationFunctionality Large-Scale HepaRG Expansion

The cells are cultured using Hyperflasks™ (Coming) as follows. Themedium is refreshed every three days. After a 2 weeks proliferationphase, at confluence, the cultures are washed twice in phosphatebuffered saline and then detached by 15 min incubation in 60ml/Hyperflask™ of Accutase™ and Accumax™ (both from Innovative CellTechnologies Inc.) in a 4:1 ratio at 37° C. The cells are harvested fromthe Hyperflask™ and three times washed with HepaRG medium bycentrifugation at 4° C. and 3×50g. Approximately 1.2 ml of cell pelletis harvested from one Hyperflask™. The harvested cells are used forpassaging at a split ratio of 1:6 for further expansion or for loadinginto the AMC-BAL bioreactor. Viability is determined by trypan blueexclusion test: cells are only used to charge the AMC-BAL biroreactor iftheir viability exceeds 95%.

AMC-BAL Bioreactor Charging and Culturing

One litre of HepaRG medium containing 75 ml of viable HepaRG cells isinjected under sterile conditions into an AMC-BAL bioreactor (cf.Flendrig et al.¹⁵) through three loading ports. The bioreactor is thenplaced in a culture cabinet at 37° C. and oxygenated with culture gas(55%N₂/40%O₂/5%CO₂) at a flow rate of 150 mL/min during a 3-hourattachment period. To ensure optimal cell attachment and an even celldistribution, bioreactors are rotated 340° (back and forth) along thelongitudinal axis at 1 revolution/min After this attachment period, deadand unattached cells are removed by flushing 500 mL of fresh culturemedium through the bioreactor at 150 mL/min From then on bioreactors areperfused with 1.5 L recirculating culture medium at 150 mL/min Themedium is refreshed every three days. The bioreactors are cultured inHepaRG medium for 14 days. In that period, the cells will undergo aboutone population doubling. The bioreactors can be continued to be culturedfor at least another 3 weeks without changes in phenotype. At least oneweek prior to clinical application, 1 mM CG (Sigma) is added to themedia to enhance the urea cycle. The bioreactors are tested everyculture week and prior to clinical application for sterility andfunction. Laboratory model AMC-BAL bioreactors, e.g. of a differentscale, are produced in essentially the same way as described above.These laboratory model bioreactors are used e.g. for clinical testing inanimal models, such as rats with acute liver failure.

Sterility Test

Culture medium perfused for three days through the bioreactor isassessed for sterility by the tests summarized in the below Table 5.

TABLE 5 Sterility tests for bioartificial liver application ACCEPTANCETEST METHOD CRITERIA Bacteria Bacterial DNA (PCR) Negative Standardculture <5 CFU/ml Mycoplasma DNA PCR negative Fungi Standard culture <5CFU/ml

Tests of AMC-BAL Functionality

The purpose of this test is to monitor the bioreactor culture and toassess the quality of the hepatocytes in the bioreactors prior toclinical application. These tests are performed once a week.

Firstly, small pieces of matrix (T-bags; 3 per time point) are withdrawnfrom the bioreactor. These matrices allow the harvesting of cells fromthe bioreactor, see Poyck et al.¹⁶ The matrices are collected in 1 ml ofTrizol (GibcoBRL). Subsequently RNA is isolated according to theinstructions of the manufacturer. Next, levels of mRNAs of hepatic genesand 18S rRNA are quantified by RT-PCR.

Secondly, oxygen consumption is determined by measuring the decrease inoxygen tension during the first 15 min after closure of the oxygensupply to the bioreactor as described by Van de Kerkhove et al ²⁰.

Thirdly, the culture medium is replaced by 1500 ml test medium. Thehepatocytes in the AMC-BAL bioreactors are exposed to a recirculatingiso-osmotic test medium for 24 hours, containing HepaRG medium (−DMSO)with different substrates (1.5 mM ¹⁵NH4Cl, 125 μM testosterone and 2.75mM galactose) together with 2 mM lactate. At first 700 ml test medium isflushed through the bioreactor followed by recirculation of 800 ml testmedium during 24 hours. At t=0 (test medium before connection to thebioreactor), t=30′, 60′, 120′, 180′, 8 hrs and 24 hrs a 1 mL sample istaken and analysed for substrate (ammonia, testosterone, lactate,glucose) and product (urea, apoA1, albumin) concentration as well ascell damage (AST, aspartate-amino transferase and LDH, lactatedehydrogenase). From the changes in concentration an activity per hourand per billion cells seeded is calculated. Quality of the cell-loadedbioreactors, measured by hepatocyte function at the start of thetreatment, is compared with values of bioreactors charged with primaryhepatocytes that have been tested extensively in vitro and in vivo inthe past.

Table 6 shows the comparison between the HepaRG cells cultured inmonolayer for 30 days with/without 2% DMSO added during the last 15 days(method according to patent US 2005/0064594) and HepaRG cells culturedin a laboratory model AMC-BAL bioreactors (10 mL internal volume) loadedwith a suspension containing 2 mL HepaRG cell pellet and cultured for 13days in absence of DMSO, but in presence of 1 mM CG.

These data indicate that the new culture method (in AMC-BAL bioreactor+CG −DMSO) improves hepatic differentiation markedly compared to theinitial culture conditions (monolayer, −CG, either with or withoutDMSO); the urea production, conversion of ammonia into urea, andtranscript levels of CPS, ArgI, GS, HNF4 and Alb are significantlyimproved.

TABLE 6 Effects of bioreactor culturing including CG addition andabsence of DMSO on the functionality of HepaRG cells, compared tomonolayer cultures. Functionality per culture system % improvement bybioreactor versus Monolayer + DMSO − Monolayer − Bioreactor −Monolayer + DMSO − Monolayer − DMSO − CG DMSO − CG DMSO − CG CG CGAmmonia elimination (nmol/h/mg 110 ± 49  66.0 ± 28.0 143 ± 105 134 199 *protein) Urea production (nmol/h/mg protein) 7.0 ± 7.0 5.0 ± 2.0 13.1 ±1.2  187 * 262 * 15N urea enrichment (% of total 16.0 ± 8.4  6.0 ± 4.725.1 ± 3.7  157 * 418 * urea) Lactate consumption (nmol/h/mg productionproduction 42.1 ± 14.7 conversion * conversion * protein) ApoA1synthesis (ug/h/mg protein) 1.2 ± 1.1 0.78 ± 0.50 1.2 ± 0.9 113 154 CPSmRNA level (% of in vivo) 1.1 ± 0.4 30.5 ± 19.1 44.1 ± 32.2 4000 *  145ArgI mRNA level (% of in vivo) 4.6 ± 3.7 14.8 ± 11.3 22.9 ± 10.4 460 *155 GS mRNA level (% of in vivo) 90.1 ± 29.1  214 ± 66.1 643 ± 395 714300 * ArgII mRNA level (% of in vivo) 718 ± 463 570 ± 364 305 ± 143 42 54 HNF4 mRNA level (% of in vivo) 60.8 ± 21.1 88.3 ± 89   386 ± 216198 * 187 * PXR mRNA level (% of in vivo) 41.7 ± 19.2 44.1 ± 16.4 82.5 ±68.9 633 437 CYP2B6 mRNA level (% of in vivo) 60.7 ± 30.0 3.5 ± 1.3 30.2± 15.9 49 863 * CYP3A4 mRNA level (% of in vivo) 68.9 ± 47.2 9.6 ± 8.2 102 ± 49.4 148 1063 *  Alb mRNA level (% of in vivo) 22.7 ± 4.4  66.4 ±24.3 72.1 ± 26.0 313 * 109 (CPS, carbamoyl phosphate synthetase; ArgI,arginase I, GS, glutamine synthetase; ArgII, Arginase II; HNF4,hepatocyte nuclear factor 4; PXR, pregnaneX receptor; CYP2B6, cytochromeP450 2B6; CYP3A4, cytochrome P450 3A4; Alb, albumin) * = P < 0.05.

EXAMPLE 3 Operating Procedure of Application of the Bioartificial Liver

The charged and tested AMC-BAL bioreactor, as described in example 1, isflushed by an electrolyte solution (Schiwa) to wash out test- andculture medium and transported while stored on ice to the medical centrein demand of the bioreactor. The charged bioreactor is connected to thepatient within 4 hours.

Extra-corporeal hemodialysis and plasmapheresis are standard proceduresat Intensive Cares (IC). The BAL system (see FIG. 1) consists of aplasma circuit including the bioreactor and a blood circuitincorporating a plasma-separator. Blood is pumped (50 ml/min) from theveno-venous or arterio-venous catheters to the plasma separator(Braun-Carex Diapact CRRT). This system enables continuousplasmapheresis for a period of 12 hours, after which the disposableneeds to be replaced. The plasmafilter is Hemaplex BT 900/A 0.2 m². Theplasma perfuses then through the bioreactor.

Between the bioreactor and the return-plasma line to the patient twofilters are inserted: one filter is a polyester cell filter (leucocytepolyester filter) and the second one serially placed is a 0.2 micronfilter, surface 0.1 m², to remove cells or liver cell debris. A highflow plasma loop (normally at 150 ml/min) is introduced to control theplasma flow inside the bioreactors. After recirculation through thebioreactor, the treated plasma is reunited with the blood cells from theplasma-separator and returned to the patient. The BAL-system has a totalextracorporeal volume of 950 ml. The BAL-Incubator and tubing sets arecustom made of standard dialysis components (RAND, Mirandola, Italy). Acomputerised pressure monitoring system is included to check thecondition of the two plasma filters regarding membrane plugging andfouling. Additional safety features involved controls for gas flow,temperature, and balanced plasma exchange.

The used Diapact CRRT plasmapheresis device has been qualified accordingto IEC 513, IEC 529, EN 60601-1, prEN 1441, IEC 601-1-4. IEC 601-2-16,EN 60601-1-2.

The plasmapheresis device is mounted according to the manufacturersinstructions with the recommended sterile tubing set and hollow fibreplasmapheresis filter Hemaplex BT 900/A 0.2 m².

The system is primed with physiological saline and air bubbles areremoved.

The patient is connected to the monitor: ECG and arterial pressure aremonitored automatically. After iodation of the skin a veno-venouscatheter is inserted by the physician in a cubital or femoral vein. Ifno venous access can be obtained the femoral artery will be used.

Plasma flow from the patient is 30-40 ml/min Plasma flow across thebioreactor will be normally about 150 ml/min

If plasmapheresis by plasma filtration (mostly 0.5 micron pore diameter)is used no extra filter is needed in the plasma inflow circuit to thebioreactor (see appendix 1). Sufficient anticoagulation will be obtainedby infusion of low molecular weight heparin. After connecting thepatient to the plasmapheresis circuit, an observation period of at leastone hour of stable haemodynamics is required before the bioreactor isinserted in the circuit. Plasmapheresis is continuous for periods ofmaximally 12 hours, interrupted by a plasmapheresis free period ofanother 12 hours. If necessary the BAL is refreshed by a new one.

REFERENCES

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1. A cell culture comprising differentiated cells from a humanhepatocyte cell line in a suitable culture medium, wherein saiddifferentiated cells have constitutive liver-specific metabolicactivity, the cell culture being obtained by a process comprising:selecting a human hepatocyte cell line; a phase of cell proliferationcomprising culturing cells of said human hepatocyte cell line in asuitable culture medium, preferably comprising serum, hormones,growth-factors and antibiotics; loading viable cells to an amount of atleast 5% of normal liver mass in a bioreactor comprising a threedimensional support matrix, and allowing the cells to attach to saidmatrix; an expansion or proliferation phase comprising culturing thecells loaded in the bioreactor in a suitable culture medium, preferablycomprising serum, hormones, growth-factors and antibiotics, to achieveat least one cell population doubling; and a phase of celldifferentiation comprising culturing the cells in a culture mediumcomprising serum, hormones, growth factors and antibiotics.
 2. The cellculture according to claim 1, wherein the cell differentiation phaselasts at least 7 days.
 3. The cell culture according to claim 1, whereinthe three dimensional support matrix comprises a porous sheet or mat, afiber network matrix, an open-pore foam, or a semi-solid material. 4.The cell culture according to claim 1, wherein said human hepatocytecell line is HepaRG, deposited at the Collection Nationale de Culturesde Microorganismes, Institut Pasteur, Accession No. 1-2652.
 5. The cellculture according to claim 1, wherein the phase of cell differentiationcomprises culturing the cells in a suitable culture medium comprising atleast 0.5 mM carbamoyl glutamate.
 6. The cell culture according to claim1, wherein the phase of cell differentiation comprises culturing thecells in a suitable culture medium that is substantially free of DMSO.7. A bioreactor, comprising a chamber or vessel holding a matrix loadedwith the cell culture of claim 1, the bioreactor further comprisingmeans for oxygenating the matrix.
 8. A bioartificial liver device,comprising the bioreactor of claim 7, a liquid circuit, a plasmaseparator or plasmapheresis unit, and one or more pumps.
 9. A method ofproducing the bioreactor of claim 7, comprising the steps of: a phase ofcell proliferation comprising culturing cells of a human hepatocyte cellline in a suitable culture medium, preferably comprising serum,hormones, growth-factors and antibiotics; loading viable cells to anamount of at least 5% of normal liver mass in a bioreactor comprising athree dimensional support matrix, and allowing the cells to attach tosaid matrix; an expansion or proliferation phase comprising culturingthe cells in a suitable culture medium, preferably comprising serum,hormones, growth-factors and antibiotics, to achieve at least one cellpopulation doubling; and a phase of cell differentiation comprisingculturing the cells in a culture medium comprising serum, hormones,growth factors and antibiotics.
 10. The method according to claim 9,wherein said human hepatocyte cell line is HepaRG, deposited at theCollection Nationale de Cultures de Microorganismes, Institut Pasteur,Accession No. 1-2652.
 11. The method according to claim 9, wherein thethree dimensional support matrix comprises a porous sheet or mat, afiber network matrix, an open-pore foam, or a semi-solid material. 12.The method according to claim 9, wherein the culture medium used in thecell differentiation phase comprises at least 0.5 mM carbamoylglutamate.
 13. The method according to claim 9, wherein the celldifferentiation phase lasts at least 7 days.
 14. The method according toclaim 9, wherein the culture medium is substantially free of DMSO.
 15. Amethod of treating a subject suffering from acute liver failure,end-stage liver disease, or acute-on-chronic liver disease, the methodcomprising extra-corporeal liver support by perfusing the subject'splasma through the bioreactor of claim
 12. 16. The method according toclaim 15, wherein the method does not comprise any steps in whichcarbamoyl glutamate is administered to the bioreactor and/or to thesubject's plasma.
 17. A process of producing a cell culture comprising:selecting a human hepatocyte cell line; a phase of cell proliferationcomprising culturing cells of said human hepatocyte cell line in asuitable culture medium, preferably comprising serum, hormones,growth-factors and antibiotics; loading viable cells to an amount of atleast 5% of normal liver mass in a bioreactor comprising a threedimensional support matrix, and allowing the cells to attach to saidmatrix; an expansion or proliferation phase comprising culturing thecells loaded in the bioreactor in a suitable culture medium, preferablycomprising serum, hormones, growth-factors and antibiotics, to achieveat least one cell population doubling; and a phase of celldifferentiation comprising culturing the cells in a culture mediumcomprising serum, hormones, growth factors and antibiotics.
 18. Theprocess according to claim 17, wherein said human hepatocyte cell lineis HepaRG, deposited at the Collection Nationale de Cultures deMicroorganismes, Institut Pasteur, Accession No. 1-2652.
 19. The processaccording to claim 17, wherein the cell differentiation phase lasts atleast 7 days.
 20. The process according to claim 17, wherein the culturemedium is substantially free of DMSO.
 21. The process according to claim17, wherein the three dimensional support matrix comprises a poroussheet or mat, a fiber network matrix, an open-pore foam, or a semi-solidmaterial.
 22. The process according to claim 17, wherein the phase ofcell differentiation comprises culturing the cells in a suitable culturemedium comprising at least 0.5 mM carbamoyl glutamate.